Please make distinction for weeks more clear. Also include data/pics from virtual -UM
12/6/13 Enzyme Assay and Inhibition Assay
Inhibition Assay Absorbance Data:
Fig 79. Inhibition assay of YopH from inhibitor 5250098 of the Chem Bridge Library
Fig 78. Inhibition assay of YopH from inhibitor 5250098 of the Chem Bridge Library (run 1)
Enzyme Assay Data:
Fig 77. Enzyme Assay results graph of enzyme concentrations vs. absorbance measured by the spectrophotometer.
12/5/13
Fig 76. Characterization poly-acrylamide page gel of YopH. Lane 1: DNA ladder, Lane 2: Cell lysate before induction, Lane 3: cell lysate after induction, Lane 4: soluble fraction, Lane 5: flow through, Lane 6: wash, Lane 7: Elution 1, Lane 8: Elution 2
FIG 75. Elution 2 of YopH after purification.
Fig 74. Elution 1 of YopH after purification.
11/20/13
Expression of YopH
Fig 73. YopH, LB, and Kan solution after expression resuspended pellet in lysis buffer.
weight of pellet: 1.47g
Measuring OD values - 23uL of total culture
Time Abs at 600nm
9:53 .086
10:29 .075
11:00 .099
12:09 .28
12:34 .36
12:46 .385
1:07 .47
1:12 .47
1:20 .52
Ligand Prep protocol was completed on 11/14/13, in order to make the ligands ready to be screened. On 11/1/5/13, virtual screening of our target was begun and is currently being run through GOLD.
11/15/13
Fig 72. DNA sequencing results from clone 2 of PP2A in T. brucei.
Obviously, no cloning whatsoever happened. The vector may have simply closed up. No results were close enough for any match for comparison because the sequence was simply:
NNNNNNNNNNNNNNNNNNNNNNNNNNNANNNNNGGTGGTGGTGGNGNNNCTCTANGNNNNNNNNTGNNGANNNNNNNNNANNNNNNNNNNNNNNNNNNNNNNGNNNNNNGAANNNNNNNNNNNNNNNNNNNNANACNNNTTNTNNNNNANNNGT
Fig 71. DNA sequencing results from clone 1 of PP2A in T. brucei.
As evident, only the ends of the gene were cloned with nothing in the center. I have to talk to Dr. B about this as to why still.
Fig 70. Master plate of single colony picked from the original transformation of 2ul pNIC-bsa4 and 4ul PP2A.
Fig 69. Regrown transformation plate of 2ul vector, pNIC-bsa4, and 4ul insert, PP2A, resulting in the same as below of streaking with no true colonies.
Fig 68. Regrown transformation plate of 3ul vector, pNIC-bsa4, and 6ul insert, PP2A.
This was regrown by streaking a pipette tip from the overgrown plate onto this plate. Obviously, it continued to grow just on the areas streaked, resulting in no solo colonies.
Fig 67. Miniprep concentration of trial 2 of pNIC-bsa4 and PP2A.
Trial 2 is from the same colony as trial 1, however, it is just a regrown colony since trial 1 was left in the shaking incubator for an extra day.
Fig 66. Miniprep concentration of trial 1 of pNIC-bsa4 and PP2A. I believe the reason why the concentration was so high is due to the fact that it was left in the shaking incubator for an extra day. However, the nanodrop reading looks to be normal, just highly concentrated.
Fig 65. PCR cleanup concentration of sample 4 of pNIC-bsa4 and PP2A
Fig 64. PCR cleanup concentration of sample 3 of pNIC-bsa4 and PP2A
Fig 63. PCR clean up concentration of sample 2 of pNIC-bsa4 and PP2A
Fig 62. PCR cleanup concentration of sample 1 of pNIC-bsa4 and PP2A
10/29/13
Where ligand is from?
Organism
Protein
Name
Control
XLogP
Molecular weight
H-donor
H-acceptor
PDB -> pubChem
Homo sapiens
4LAC
adenosine 5'-O-(3-thiotriphosphate)
positive
CID_440317
PubChem
4-aminophenylphosphate (pNPP)
positive
CID_77949
PubChem
Homo Sapiens
2NPP
calyculin A
positive
CID_5311365
1.8
1009.169702 g/mol
8
18
PubChem
Homo Sapiens
2NPP
fostriecin
positive
CID_6436285
-0.5
430.386042 g/mol
5
9
PubChem
Homo Sapiens
2NPP
microcystin
positive
CID_445434
2.3
995.17166 g/mol
10
13
PubChem
Homo Sapiens
2NPP
okadaic acid
positive
CID_446512
3.4
805.00292 g/mol
5
13
PubChem
Homo Sapiens
2NPP
nodularin
positive
CID_6437077
1.7
824.9627 g/mol
8
11
PuChem
Homo Sapiens
caffeic acid phenethyl ester
positive
CID_5281787
4.2
284.30654 g/mol
2
4
PubChem
Homo Sapiens
glautamate
positive
CID_33032
-3.7
47.12926 g/mol
3
5
PubChem
Homo Sapiens
endothall
positive
CID_3225
-0.5
186.162 g/mol
2
5
Mesalazine
positive
CID_4075
1.3
153.13538 g/mol
3
4
PubChem
Asprin
ACETYLSALICYLIC ACID
negative
CID_2244
1.2
180.15742 g/mol
1
4
Chembridge
negative
5150705
3.43
516 g/mol
6
10
Chembridge
negative
5224197
3.14
486 g/mol
6
10
Chembridge
negative
5127723
2.74
401 g/mol
5
10
Chembridge
negative
7845952
3.41
493 g/mol
5
8
Fig 62. Chart of all the postive and negative control ligands for virtual screening for PP2A.
As one can see, the plates did not grow as clean of colonies as intended. In an effort to get around this, I decided to swipe some of the bacteria from each plate and spread it out on new plates (respectively), in an effort to regrow the bacteria in better looking colonies. However, one colony from the 2ul/4ul ratio was picked and chosen to make a master plate out of.
Fig 61. Transformation plate of 3ul vector (pNIC-Bsa4) and 6ul insert (protein phosphatase 2A gene).
Fig 60. Transformation plate of 2ul vector (pNIC-Bsa4) and 4ul insert (protein phosphatase 2A gene).
10/24/13
After some pretty low concentrations resulting from PCR cleanup, I decided to only go ahead and use sample 4 for transformation.
Fig 59. PCR cleanup concentration after PCR squared of protein phosphatase 2A gene, sample 4.
Fig 58. PCR cleanup concentration after PCR squared of protein phosphatase 2A gene, sample 3.
Fig 57. PCR cleanup concentration after PCR squared of protein phosphatase 2A gene, sample 2.
Fig 56. PCR cleanup concentration after PCR squared of protein phosphatase 2A gene, sample 1.
Fig 55. Virtually cut vector of pNIC-Bsa4, emphasizing where bands should be seen if cut correctly. These bands represent where in the pNic-Bsa4 that the genome is cut, allowing tail primers of the insert (gene) attach on, where the cut out sequence would be.
Fig 54. Cut accepting vector of pNIC-Bsa4 run on a gel to ensure the cuts happened and happened in the right location.
PCR squared was completely again, following up with an unsuccessful gel extraction. More accepting vector was created for our next rounds of cloning.
Virtual screening with control ligands was run. More ligands must be found and ran on GOLD.
Where ligand is from
Species
Protein
Name
Control
PDB -> pubChem
Homo sapien
4LAC
adenosine 5'-O-(3-thiotriphosphate)
positive
CID_440317
PubChem
4-aminophenylphosphate (pNPP)
positive
CID_77949
PubChem
Asprin
ACETYLSALICYLIC ACID
negative
CID_2244
Week 5 &6 Nice Job keeping your work up to date. Good captions but please try to add a brief analysis after new data. Thank you. -Max 10/07/2013 Homology Model: A homology model was created since our protein, protein phosphatase 2A, does not have one.
Fig 53. Molprobity Multi-criterion chart of the newly created homology model for PP2A created using 2nppF.
Fig 52. Molprobity Multi-criterion chart of chain F of 2npp
Chain F of 2npp has a 71% Identity and 84% Positives Top hit from SWISS-MODEL test is chain F of 2NPP. The amino acid sequence for 2nppF is: MDEKVFTKELDQWIEQLNECKQLSESQVKSLCEKAKEILTKESNVQEVRCPVTVCGDVHGQFHDLMELFRIGGKSPDTNY LFMGDYVDRGYYSVETVTLLVALKVRYRERITILRGNHESRQITQVYGFYDECLRKYGNANVWKYFTDLFDYLPLTALVD GQIFCLHGGLSPSIDTLDHIRALDRLQEVPHEGPMCDLLWSDPDDRGGWGISPRGAGYTFGQDISETFNHANGLTLVSRA HQLVMEGYNWCHDRNVVTIFSAPNYCYRCGNQAAIMELDDTLKYSFLQFDPAPRRGEPHVTRRTPDYFL
Fig 51. SWISS-MODEL homology test top results.
Fig 50. BLASTp results to find a homology model for PP2A
Homology Model: A homology model was created since our protein, protein phosphatase 2A, does not have one.
Nanodrop results of the second round of cloning look much more promising than the first round. Results should be in in a day or two. Fig 49. Nanodrop concentration of tube 8 after miniprep
Fig 48. Nanodrop concentration of tube 7 after miniprep
Fig 47. Nanodrop concentration of tube 6 after miniprep
Fig 46. Nanodrop concentration of tube 5 after miniprep.
Fig 45. Nanodrop concentration of tube 4 after miniprep.
Fig 44. Nanodrop concentration of tube 3 after miniprep.
Fig 43. Nanodrop concentration of tube 2 after miniprep.
Fig 42. Nanodrop concentration of tube 1 after miniprep.
Fig 41. Master plate used in second round of cloning, 2 ul of accepting vector and 4 ul of insert
Fig 40. Secondary PCR of T. brucei - Lane 1-4: Renee and Tony's samples, Lane 5: blank, Lane 6: 100 base pair ladder, Lane 7: primary PCR, Lane 8: secondary PCR sample A, Lane 9: secondary PCR sample B.
None of the results received were completely successful. Tube 3 was the closest but there was one deletion which will not let us precede with this sample. A second round of cloning will soon begin. Fig. Tube 3 of master plate B cloning results, Query cover 99%, Identity 98%, closest sample we had, yet there was a deletion in the second row.
Fig. Tube 7 of master plate tube B cloning results, Query cover 93%, Identity 96%
Week 3 & 4 Kevin - ok good work on the colonies. Show if these sequences were positive or not from Sequencing. - DR. B 101013 Fig 39. Nanodrop concentration of tube 1 after miniprep
Fig 38. Nanodrop concentration of tube 2 after miniprep. Fig 37. Nanodrop concentration of tube 3 after miniprep. Fig 36. Nanodrop concentration of tube 4 after miniprep. Fig 35. Nanodrop concentration of tube 5 after miniprep. Fig 34. Nanodrop concentration of tube 6 after miniprep. Fig 33. Nanodrop concentration of tube 7 after miniprep. Fig 32. Nanodrop concentration of tube 8 after miniprep. Fig 31. Master plate with transformation mixture from tube B (3ul of accepting vector and 6 ul of insert)
Fig 30. Master plate with transformation mixture from tube A (2ul of accepting vector and 4 ul of insert)
Master plates were grown with 8 colonies from the transformation plate in 5mL LB and 5 uL kanamacyn. We also started a back up master plate in case these results were not successful.
Fig 29. Successful cut vector of pNIC-BSA4 gel. After taking a picture without the rig, the strange imaging at the bottom of the gel disappeared.
Fig 28. NEBcutter, virtual gel of the cut vector pNIC-BSA4.
Fig 27. PCR clean up for the 8 samples run off of secondary PCR
Fall 2013 - Week 1 & 2 Kevin, need some more results here (PCR's etc.) Also include an image of PyMol refresher. Each week should have some image ideally. - Dr. B 090913
This week in VDS, I completed a PyMol refresher. Also, I completed the preparation of pNIC-Bsa4 as accepting vector. This RE Digest is necessary for cutting the pNIC so that my gene can insert into the DNA before cloning. Fig 26. PyMol of 2H2Q PDB. Selected substrates NAP and DU shown as red. Polar contacts between atoms shown as black. Hydrophobic residues shown as yellow; ionic residues shown as blue; polar residues shown as orange. All other atoms colored by element with carbon as green.
Fig 25. Alignment of 1U72 and 3CL9 on PyMol. Active sites around MTX (red) are pink for 3CL9 and navy for 1U72. In all, 1U72 is colored by element with carbon as green and 3CL9 with carbon as cyan. Week 6 Kevin - good conc of pNIC. SHow some more of your Overlap and Secondary PCR.s - Dr. B 071713
Ordered new correct primers: Fig 24. Successful Secondary PCR of T. Brucei tail primers Lane 1: Skip, Lane 2: 100 bp ladder, Lane 3: Sample 1, Lane 4: Sample 2, Lane 5: Sample 3, Lane 6: Sample 4, Lane 7: Sample 5, Lane 8: Sample 6, Lane 9: Sample 7, Lane 10: Sample 8
Sample cycle: 98 for 30s, [98 for 10s, for 30s, 72 for 30s] 25
Cycles, 72 for 2 minutes, 4 for forever
Fig 22 and 23. Measurement 1 and 2 of pUC19 from the nanodrop after midiprep
Fig 21. Secondary PCR gel of T. Brucei. Lane 1: 100 bp ladder, Lane 2: Sample 12, Lane 3: Sample 13, Lane 4: Sample 14, Lane 5: Sample 15, Lane 6: Sample 16, Lane 7: Sample 17, Lane 8: Sample 18, Lane 9: Sample 19, Lane 10: Not our sample
Sample 12: 98 degrees for 30s, [98 for 10s, 72 for 30s, 72 for 30s] 25 cycles, 72 for 2 min, 4 for forever.
Sample 13: 98 degrees for 30s, [98 for 10s, 70.5 for 30s, 72 for 30s] 25 cycles, 72 for 2 min, 4 for forever.
Sample 14: 98 degrees for 30s, [98 for 10s, 68.1 for 30s, 72 for 30s] 25 cycles, 72 for 2 min, 4 for forever.
Sample 15: 98 degrees for 30s, [98 for 10s, 64.5 for 30s, 72 for 30s] 25 cycles, 72 for 2 min, 4 for forever.
Sample 16: 98 degrees for 30s, [98 for 10s, 59.9 for 30s, 72 for 30s] 25 cycles, 72 for 2 min, 4 for forever.
Sample 17: 98 degrees for 30s, [98 for 10s, 56.5 for 30s, 72 for 30s] 25 cycles, 72 for 2 min, 4 for forever.
Sample 18: 98 degrees for 30s, [98 for 10s, 53.8 for 30s, 72 for 30s] 25 cycles, 72 for 2 min, 4 for forever.
Sample 19: 98 degrees for 30s, [98 for 10s, 52 for 30s, 72 for 30s] 25 cycles, 72 for 2 min, 4 for forever.
Fig 20. Secondary PCR gel of T. Brucei. Lane 1: 100 bp ladder, Lane 2: Sample 6, Lane 3: Sample 7, Lane 4: Sample 8, Lane 5: Sample 9, Lane 6: Sample 10, Lane 7: Sample 11
Sample 11: 98 degrees for 30 s, [98 for 15s, 66.2 for 30s, 72 for 10s] 25 cycles, 72 for 2 min, 4 for forever.
Sample 10: 98 degrees for 30 s, [98 for 15s, 67 for 30s, 72 for 10s] 25 cycles, 72 for 2 min, 4 for forever.
Sample 9: 98 degrees for 30 s, [98 for 15s, 68.2 for 30s, 72 for 10s] 25 cycles, 72 for 2 min, 4 for forever.
Sample 8: 98 degrees for 30 s, [98 for 15s, 69.1 for 30s, 72 for 10s] 25 cycles, 72 for 2 min, 4 for forever.
Sample 7: 98 degrees for 30 s, [98 for 15s, 69.7 for 30s, 72 for 10s] 25 cycles, 72 for 2 min, 4 for forever.
Sample 6: 98 degrees for 30 s, [98 for 15s, 70 for 30s, 72 for 10s] 25 cycles, 72 for 2 min, 4 for forever.
Fig 19. Secondary PCR gel of T. Brucei
Lane 1: blank, Lane 2: 100 bp ladder, Lane 3: Sample 3, Lane 4: Sample 4.
Sample 4: 98 degrees for 30 s, [98 for 15s, 60 for 30s, 72 for 30s] 20 cycles, 72 for 2 min, 4 for forever.
Sample 3: 98 degrees for 30 s, [98 for 15s, 60 for 10s, 72 for 30s] 20 cycles, 72 for 2 min, 4 for forever.
Fig 18. PCR gel of T. Brucei; Lane 1 & 2 blank, Lane 3: 100 bp ladder, Lane 4: Primary PCR, Lane 5-8: Jackie's sample, Lane 9: Sample 1, Lane 10: Sample 2.
Sample 2: 98 degrees for 30 s, [98 for 30s, 65 for 30s, 72 for 30s] 20 cycles, 72 for 2 min, 4 for forever.
Sample 1: 98 degrees for 30 s, [98 for 10s, 65 for 20s, 72 for 30s] 20 cycles, 72 for 2 min, 4 for forever.
Fig 17. Nanodrop trial 1 and 2 after midiprep of pNIC-BSA4. Determination of concentration is 72.2 ng/uL.
Fig 16. Graph of FtHap FPLC of ptp1b
Fig 15. Gel of PCR overlap of T. Bruci
Lane 1: KB ladder
Lane 2: Primary PCR
Lane 3: Secondary PCR Failure due to using the ladder instead of the blue dye in samples. Will run using same samples next.
Fig 14. PCR gel of pNIC-Bsa4.
Lane 1 – 1 KB ladder
Lane 2 – lowest concentration
Lane 3 – medium concentration
Lane 4 – highest concentration
Lane 5 – control, no template
Fig 13. Gel of PCR of pGBR22 purple protein. Lane 1: 1 KB ladder, Lane 2: Sample A (1:1000 dilution of DNA template, 1 microliter), Lane 3: Sample B (1:1000 dilution of template, 10 microliters), Lane 4: Sample C (1:100 dilution of template, 10 microliters), Lane 5: Sample D (control, no DNA template).
Template seen through gel in lane 5, the control, may be due to leakage of the wells.
Fig 12. RE Digest of pGBR22 plasmid with restriction enzymes EcoRI and PvuII. Lane 1: skipped, Lane 2: 1 KB ladder, Lane 3: Uncut plasmid, Lane 4: EcoRI digestion, Lane 5: PvuII digestion, Lane 6: EcoRI and PvuII digestion, Lane 7: 1 KB ladder, Lane 8: EcoRI digestion, Lane 9: PvuII digestion, Lane 10: EcoRI and PvuII digestion.
Fig 9. Agarose gel PCR of pmCherry into pGBR22 plasmid
Lane 1 - 1KB ladder, Lane 2 - sample 1, Lane 3 - sample 2, Lane 4 - sample 3, Lane 5 - sample 4, Lane 6 - sample 5, Lane 7 - sample 6, Lane 8 - sample 7, Lane 9 - sample 8 Agarose gel
Fig 8. 1KB DNA ladder used from NEB used in running gels.
Fig 7. Agarose gel of pGBR22 plasmid, primers M13 forward and reverse:
Lane 1 – 1KB latter
Lane 2 – Sample A
Lane 3 – Sample B
Lane 4 – Sample C
Lane 5 – Sample D
Fig 6. Nanodrop measurement 2 of pNIC-Bsa4 plasmid at 230 nm.
Fig 5. Nanodrop measurement 1 of pNIC-Bsa4 plasmid + FABI at 230 nm.
Fig 4. Transformation of E. coli with 5ng pNIC-BSA4, NEB 5 alpha grown on LB and Kan agar plates.
Fig. 3 Transformation of E. coli with 25ng pNIC-BSA4, NEB 5 alpha grown on LB and Kan agar plates.
Fig. 2 Transformation of E. coli with 25ng pNIC-BSA4, NEB 5 alpha grown on LB and Kan agar plates.
Fig 1. Nanodrop spectrophotometer measurements 1 and 2 of pGBR22 plasmid blanked using TE buffer.
Kevin - update your page and properly annotate everything. All of your results for the last 2 weeks shoudl be here. Also repeat this PCR. What was the PCR of? - Dr. B
Kevin - good reverse chronological order, but segment your results by each week (We are now on Week 6). Also put dates for each image (roughly) - Dr. B 070913
12/6/13
Enzyme Assay and Inhibition Assay
Inhibition Assay Absorbance Data:
Fig 79. Inhibition assay of YopH from inhibitor 5250098 of the Chem Bridge Library
Fig 78. Inhibition assay of YopH from inhibitor 5250098 of the Chem Bridge Library (run 1)
Enzyme Assay Data:
Fig 77. Enzyme Assay results graph of enzyme concentrations vs. absorbance measured by the spectrophotometer.
12/5/13
Fig 76. Characterization poly-acrylamide page gel of YopH. Lane 1: DNA ladder, Lane 2: Cell lysate before induction, Lane 3: cell lysate after induction, Lane 4: soluble fraction, Lane 5: flow through, Lane 6: wash, Lane 7: Elution 1, Lane 8: Elution 2
FIG 75. Elution 2 of YopH after purification.
Fig 74. Elution 1 of YopH after purification.
11/20/13
Expression of YopH
Fig 73. YopH, LB, and Kan solution after expression resuspended pellet in lysis buffer.
weight of pellet: 1.47g
Measuring OD values - 23uL of total culture
Time Abs at 600nm
9:53 .086
10:29 .075
11:00 .099
12:09 .28
12:34 .36
12:46 .385
1:07 .47
1:12 .47
1:20 .52
Ligand Prep protocol was completed on 11/14/13, in order to make the ligands ready to be screened. On 11/1/5/13, virtual screening of our target was begun and is currently being run through GOLD.
11/15/13
Fig 72. DNA sequencing results from clone 2 of PP2A in T. brucei.
Obviously, no cloning whatsoever happened. The vector may have simply closed up. No results were close enough for any match for comparison because the sequence was simply:
NNNNNNNNNNNNNNNNNNNNNNNNNNNANNNNNGGTGGTGGTGGNGNNNCTCTANGNNNNNNNNTGNNGANNNNNNNNNANNNNNNNNNNNNNNNNNNNNNNGNNNNNNGAANNNNNNNNNNNNNNNNNNNNANACNNNTTNTNNNNNANNNGT
Fig 71. DNA sequencing results from clone 1 of PP2A in T. brucei.
As evident, only the ends of the gene were cloned with nothing in the center. I have to talk to Dr. B about this as to why still.
Fig 70. Master plate of single colony picked from the original transformation of 2ul pNIC-bsa4 and 4ul PP2A.
Fig 69. Regrown transformation plate of 2ul vector, pNIC-bsa4, and 4ul insert, PP2A, resulting in the same as below of streaking with no true colonies.
Fig 68. Regrown transformation plate of 3ul vector, pNIC-bsa4, and 6ul insert, PP2A.
This was regrown by streaking a pipette tip from the overgrown plate onto this plate. Obviously, it continued to grow just on the areas streaked, resulting in no solo colonies.
Fig 67. Miniprep concentration of trial 2 of pNIC-bsa4 and PP2A.
Trial 2 is from the same colony as trial 1, however, it is just a regrown colony since trial 1 was left in the shaking incubator for an extra day.
Fig 66. Miniprep concentration of trial 1 of pNIC-bsa4 and PP2A. I believe the reason why the concentration was so high is due to the fact that it was left in the shaking incubator for an extra day. However, the nanodrop reading looks to be normal, just highly concentrated.
Fig 65. PCR cleanup concentration of sample 4 of pNIC-bsa4 and PP2A
Fig 64. PCR cleanup concentration of sample 3 of pNIC-bsa4 and PP2A
Fig 63. PCR clean up concentration of sample 2 of pNIC-bsa4 and PP2A
Fig 62. PCR cleanup concentration of sample 1 of pNIC-bsa4 and PP2A
10/29/13
As one can see, the plates did not grow as clean of colonies as intended. In an effort to get around this, I decided to swipe some of the bacteria from each plate and spread it out on new plates (respectively), in an effort to regrow the bacteria in better looking colonies. However, one colony from the 2ul/4ul ratio was picked and chosen to make a master plate out of.
Fig 61. Transformation plate of 3ul vector (pNIC-Bsa4) and 6ul insert (protein phosphatase 2A gene).
Fig 60. Transformation plate of 2ul vector (pNIC-Bsa4) and 4ul insert (protein phosphatase 2A gene).
10/24/13
After some pretty low concentrations resulting from PCR cleanup, I decided to only go ahead and use sample 4 for transformation.
Fig 59. PCR cleanup concentration after PCR squared of protein phosphatase 2A gene, sample 4.
Fig 58. PCR cleanup concentration after PCR squared of protein phosphatase 2A gene, sample 3.
Fig 57. PCR cleanup concentration after PCR squared of protein phosphatase 2A gene, sample 2.
Fig 56. PCR cleanup concentration after PCR squared of protein phosphatase 2A gene, sample 1.
Fig 55. Virtually cut vector of pNIC-Bsa4, emphasizing where bands should be seen if cut correctly. These bands represent where in the pNic-Bsa4 that the genome is cut, allowing tail primers of the insert (gene) attach on, where the cut out sequence would be.
Fig 54. Cut accepting vector of pNIC-Bsa4 run on a gel to ensure the cuts happened and happened in the right location.
PCR squared was completely again, following up with an unsuccessful gel extraction. More accepting vector was created for our next rounds of cloning.
Virtual screening with control ligands was run. More ligands must be found and ran on GOLD.
Week 5 &6
Nice Job keeping your work up to date. Good captions but please try to add a brief analysis after new data. Thank you. -Max 10/07/2013
Homology Model:
A homology model was created since our protein, protein phosphatase 2A, does not have one.
Fig 53. Molprobity Multi-criterion chart of the newly created homology model for PP2A created using 2nppF.
Fig 52. Molprobity Multi-criterion chart of chain F of 2npp
Chain F of 2npp has a 71% Identity and 84% Positives
Top hit from SWISS-MODEL test is chain F of 2NPP. The amino acid sequence for 2nppF is:
MDEKVFTKELDQWIEQLNECKQLSESQVKSLCEKAKEILTKESNVQEVRCPVTVCGDVHGQFHDLMELFRIGGKSPDTNY
LFMGDYVDRGYYSVETVTLLVALKVRYRERITILRGNHESRQITQVYGFYDECLRKYGNANVWKYFTDLFDYLPLTALVD
GQIFCLHGGLSPSIDTLDHIRALDRLQEVPHEGPMCDLLWSDPDDRGGWGISPRGAGYTFGQDISETFNHANGLTLVSRA
HQLVMEGYNWCHDRNVVTIFSAPNYCYRCGNQAAIMELDDTLKYSFLQFDPAPRRGEPHVTRRTPDYFL
Fig 51. SWISS-MODEL homology test top results.
Fig 50. BLASTp results to find a homology model for PP2A
Homology Model:
A homology model was created since our protein, protein phosphatase 2A, does not have one.
Nanodrop results of the second round of cloning look much more promising than the first round. Results should be in in a day or two.
Fig 49. Nanodrop concentration of tube 8 after miniprep
Fig 48. Nanodrop concentration of tube 7 after miniprep
Fig 47. Nanodrop concentration of tube 6 after miniprep
Fig 46. Nanodrop concentration of tube 5 after miniprep.
Fig 45. Nanodrop concentration of tube 4 after miniprep.
Fig 44. Nanodrop concentration of tube 3 after miniprep.
Fig 43. Nanodrop concentration of tube 2 after miniprep.
Fig 42. Nanodrop concentration of tube 1 after miniprep.
Fig 41. Master plate used in second round of cloning, 2 ul of accepting vector and 4 ul of insert
Fig 40. Secondary PCR of T. brucei - Lane 1-4: Renee and Tony's samples, Lane 5: blank, Lane 6: 100 base pair ladder, Lane 7: primary PCR, Lane 8: secondary PCR sample A, Lane 9: secondary PCR sample B.
None of the results received were completely successful. Tube 3 was the closest but there was one deletion which will not let us precede with this sample. A second round of cloning will soon begin.
Fig. Tube 3 of master plate B cloning results, Query cover 99%, Identity 98%, closest sample we had, yet there was a deletion in the second row.
Fig. Tube 7 of master plate tube B cloning results, Query cover 93%, Identity 96%
Fig. Tube 5 cloning results, Query cover 89%, Identity 99%
Week 3 & 4
Kevin - ok good work on the colonies. Show if these sequences were positive or not from Sequencing. - DR. B 101013
Fig 39. Nanodrop concentration of tube 1 after miniprep
Fig 38. Nanodrop concentration of tube 2 after miniprep.
Fig 37. Nanodrop concentration of tube 3 after miniprep.
Fig 36. Nanodrop concentration of tube 4 after miniprep.
Fig 35. Nanodrop concentration of tube 5 after miniprep.
Fig 34. Nanodrop concentration of tube 6 after miniprep.
Fig 33. Nanodrop concentration of tube 7 after miniprep.
Fig 32. Nanodrop concentration of tube 8 after miniprep.
Fig 31. Master plate with transformation mixture from tube B (3ul of accepting vector and 6 ul of insert)
Fig 30. Master plate with transformation mixture from tube A (2ul of accepting vector and 4 ul of insert)
Master plates were grown with 8 colonies from the transformation plate in 5mL LB and 5 uL kanamacyn. We also started a back up master plate in case these results were not successful.
Fig 29. Successful cut vector of pNIC-BSA4 gel. After taking a picture without the rig, the strange imaging at the bottom of the gel disappeared.
Fig 28. NEBcutter, virtual gel of the cut vector pNIC-BSA4.
Fig 27. PCR clean up for the 8 samples run off of secondary PCR
Fall 2013 - Week 1 & 2
Kevin, need some more results here (PCR's etc.) Also include an image of PyMol refresher. Each week should have some image ideally. - Dr. B 090913
This week in VDS, I completed a PyMol refresher.
Also, I completed the preparation of pNIC-Bsa4 as accepting vector. This RE Digest is necessary for cutting the pNIC so that my gene can insert into the DNA before cloning.
Fig 26. PyMol of 2H2Q PDB. Selected substrates NAP and DU shown as red. Polar contacts between atoms shown as black. Hydrophobic residues shown as yellow; ionic residues shown as blue; polar residues shown as orange. All other atoms colored by element with carbon as green.
Fig 25. Alignment of 1U72 and 3CL9 on PyMol. Active sites around MTX (red) are pink for 3CL9 and navy for 1U72. In all, 1U72 is colored by element with carbon as green and 3CL9 with carbon as cyan.
Week 6
Kevin - good conc of pNIC. SHow some more of your Overlap and Secondary PCR.s - Dr. B 071713
Ordered new correct primers:
Fig 24. Successful Secondary PCR of T. Brucei tail primers
Lane 1: Skip, Lane 2: 100 bp ladder, Lane 3: Sample 1, Lane 4:
Sample 2, Lane 5: Sample 3, Lane 6: Sample 4, Lane 7: Sample
5, Lane 8: Sample 6, Lane 9: Sample 7, Lane 10: Sample 8
Sample cycle: 98 for 30s, [98 for 10s, for 30s, 72 for 30s] 25
Cycles, 72 for 2 minutes, 4 for forever
- Sample 1: 72°, Sample 2: 71.4°, Sample 3: 70.1°, Sample
4: 68.3°, Sample 5: 66°, Sample 6: 64.3°, Sample 7: 63°,Sample 8: 62°
Fig 22 and 23. Measurement 1 and 2 of pUC19 from the nanodrop after midiprep
Fig 21. Secondary PCR gel of T. Brucei. Lane 1: 100 bp ladder, Lane 2: Sample 12, Lane 3: Sample 13, Lane 4: Sample 14, Lane 5: Sample 15, Lane 6: Sample 16, Lane 7: Sample 17, Lane 8: Sample 18, Lane 9: Sample 19, Lane 10: Not our sample
Sample 12: 98 degrees for 30s, [98 for 10s, 72 for 30s, 72 for 30s] 25 cycles, 72 for 2 min, 4 for forever.
Sample 13: 98 degrees for 30s, [98 for 10s, 70.5 for 30s, 72 for 30s] 25 cycles, 72 for 2 min, 4 for forever.
Sample 14: 98 degrees for 30s, [98 for 10s, 68.1 for 30s, 72 for 30s] 25 cycles, 72 for 2 min, 4 for forever.
Sample 15: 98 degrees for 30s, [98 for 10s, 64.5 for 30s, 72 for 30s] 25 cycles, 72 for 2 min, 4 for forever.
Sample 16: 98 degrees for 30s, [98 for 10s, 59.9 for 30s, 72 for 30s] 25 cycles, 72 for 2 min, 4 for forever.
Sample 17: 98 degrees for 30s, [98 for 10s, 56.5 for 30s, 72 for 30s] 25 cycles, 72 for 2 min, 4 for forever.
Sample 18: 98 degrees for 30s, [98 for 10s, 53.8 for 30s, 72 for 30s] 25 cycles, 72 for 2 min, 4 for forever.
Sample 19: 98 degrees for 30s, [98 for 10s, 52 for 30s, 72 for 30s] 25 cycles, 72 for 2 min, 4 for forever.
Fig 20. Secondary PCR gel of T. Brucei. Lane 1: 100 bp ladder, Lane 2: Sample 6, Lane 3: Sample 7, Lane 4: Sample 8, Lane 5: Sample 9, Lane 6: Sample 10, Lane 7: Sample 11
Sample 11: 98 degrees for 30 s, [98 for 15s, 66.2 for 30s, 72 for 10s] 25 cycles, 72 for 2 min, 4 for forever.
Sample 10: 98 degrees for 30 s, [98 for 15s, 67 for 30s, 72 for 10s] 25 cycles, 72 for 2 min, 4 for forever.
Sample 9: 98 degrees for 30 s, [98 for 15s, 68.2 for 30s, 72 for 10s] 25 cycles, 72 for 2 min, 4 for forever.
Sample 8: 98 degrees for 30 s, [98 for 15s, 69.1 for 30s, 72 for 10s] 25 cycles, 72 for 2 min, 4 for forever.
Sample 7: 98 degrees for 30 s, [98 for 15s, 69.7 for 30s, 72 for 10s] 25 cycles, 72 for 2 min, 4 for forever.
Sample 6: 98 degrees for 30 s, [98 for 15s, 70 for 30s, 72 for 10s] 25 cycles, 72 for 2 min, 4 for forever.
Fig 19. Secondary PCR gel of T. Brucei
Lane 1: blank, Lane 2: 100 bp ladder, Lane 3: Sample 3, Lane 4: Sample 4.
Sample 4: 98 degrees for 30 s, [98 for 15s, 60 for 30s, 72 for 30s] 20 cycles, 72 for 2 min, 4 for forever.
Sample 3: 98 degrees for 30 s, [98 for 15s, 60 for 10s, 72 for 30s] 20 cycles, 72 for 2 min, 4 for forever.
Fig 18. PCR gel of T. Brucei; Lane 1 & 2 blank, Lane 3: 100 bp ladder, Lane 4: Primary PCR, Lane 5-8: Jackie's sample, Lane 9: Sample 1, Lane 10: Sample 2.
Sample 2: 98 degrees for 30 s, [98 for 30s, 65 for 30s, 72 for 30s] 20 cycles, 72 for 2 min, 4 for forever.
Sample 1: 98 degrees for 30 s, [98 for 10s, 65 for 20s, 72 for 30s] 20 cycles, 72 for 2 min, 4 for forever.
Fig 17. Nanodrop trial 1 and 2 after midiprep of pNIC-BSA4. Determination of concentration is 72.2 ng/uL.
Fig 16. Graph of FtHap FPLC of ptp1b
Fig 15. Gel of PCR overlap of T. Bruci
Lane 1: KB ladder
Lane 2: Primary PCR
Lane 3: Secondary PCR
Failure due to using the ladder instead of the blue dye in samples. Will run using same samples next.
Fig 14. PCR gel of pNIC-Bsa4.
Lane 1 – 1 KB ladder
Lane 2 – lowest concentration
Lane 3 – medium concentration
Lane 4 – highest concentration
Lane 5 – control, no template
Fig 13. Gel of PCR of pGBR22 purple protein. Lane 1: 1 KB ladder, Lane 2: Sample A (1:1000 dilution of DNA template, 1 microliter), Lane 3: Sample B (1:1000 dilution of template, 10 microliters), Lane 4: Sample C (1:100 dilution of template, 10 microliters), Lane 5: Sample D (control, no DNA template).
Template seen through gel in lane 5, the control, may be due to leakage of the wells.
Fig 12. RE Digest of pGBR22 plasmid with restriction enzymes EcoRI and PvuII. Lane 1: skipped, Lane 2: 1 KB ladder, Lane 3: Uncut plasmid, Lane 4: EcoRI digestion, Lane 5: PvuII digestion, Lane 6: EcoRI and PvuII digestion, Lane 7: 1 KB ladder, Lane 8: EcoRI digestion, Lane 9: PvuII digestion, Lane 10: EcoRI and PvuII digestion.
pNIC-Bsa4 FASTA sequence with vector in it:
TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAA
CCTGTACTTCCAATCCATGCTGTCCCTGACCCGCATCCTCCGTAAGAAGATCCCTGTTCACGAACTGGCCGGCAAGATTTCTCGTCCACCACTCCGTCCGTTTTCCGTCGTCGTTGCCAGCGATGAAAAGGGT
GGTATTGGCGACGGTGGTACTATTCCTTGGGAAATCCCAGAAGACATGCAATACTTCCGCCGTGTGACGACCAACCTGCGTGGCAAAAATGTTAAGCCGAGCCCATCTAAGCGTAACGCGGTCGTGATGGGT
CGCAAAACCTGGGATAGCCTGCCACCTAAGTTCCGTCCACTCTCTAATCGTCTGAATGTGGTTCTGTCCCGTTCTGCGACTAAAGAGCAACTGCTCGCGGGTATTCCGGACCCAATCAAGCGTGCGGAAGCT
GCTAACGACGTGGTGGCTGTTAACGGTGGCCTGGAAGATGCACTGCGTATGCTCGTAAGCAAGGAACATACCTCTAGCATCGAGACCGTGTTTTGCATCGGTGGCGGTACGATCTACAAACAGGCGCTCTGT
GCCCCTTGCGTTAATGTTCTGCAAGCGATCCACCGTACCGTTGTACGTCCGGCGTCTAACTCTTGCTCTGTTTTCTTCGACATCCCAGCGGCTGGCACCAAGACCCTCGAAGGCCTCGAACTCGTACGTGAGT
CTATTACCGATGAGCGTGTTTCTACGGGCGCTGGTGGTAAAAAGTATCAATTTGAAAAGCTCGTGCCGCGCAATAGCGAAGAAGAGCAGTATCTCAACCTCGTGGGCCGCATTATCGACGAGGGTTGCACCA
AATGTGACCGCACTGGTGTAGGTACGCGTTCCCTCTTCGGTGCTCAGATGCGTTTCTCTCTGCGTAACAACCGTCTGCCTCTGCTGACTACTAAACGCGTCTTTTGGCGTGGTGTATGTGAAGAGCTCCTCTG
GTTCCTCCGCGGTGAAACCAACGCAAAGCTCCTGTCTGATAAGGGCATCCATATTTGGGACGGTAATGGTTCCCGTGCTTTTCTCGACTCCCGTGGTCTGACCGACTACGACGAGATGGACCTCGGCCCAGT
CTATGGCTTCCAATGGCGTCACTTTGGCGCGGACTACATCTCTTGCAAAGAGGATTCCGAGGGTAAGGGCGTAGACCAAATCGCGAATATCGTCAAATCTCTCATCGAAAACCCTGATGACCGCCGTATGATTT
GCACCGCGTGGAACCCGGCTGCCCTCCCTCGCATGGCACTCCCGCCTTGCCACATGATGGCGCAATTTTACGTGTCTAATGGCGAGCTGTCCTGCATGCTCTATCAGCGCTCCTGCGATATGGGCCTGGGTG
TGCCGTTCAATATCGCGTCCTATGCGCTGCTCACGTTTCTGATGGCTAAAGCGTCTGGTCTGCGTCCGGGTGAGCTGGTACATACTCTGGGCGATGCGCACGTATACTCCAACCACGTTGAACCATGCCGTAA
ACAGCTGAAGCGTGTCCCGCGTCCATTCCCGTTTATCGTCTTCAAGCAAGATAAGGAGTTTCTGGAGGACTTTCAAGAAAGCGACATCGAGGTGATCGACTATTCTCCATACCCAGTAATCTCTATGGAAATGG
CAGTGCTCGGCGACAGCCCTGGCTACGTTATGTCCAACATCGAATTCCGTCAACTGACGCGTGGTCATAGCCCGTCCGACGAACGCGAGGCACGCCGCGTCGAGGAGGCGGGTGGTCAACTCTTCGTGAT
TGGCGGCGAACTCCGTGTAAACGGTGTCCTGAATCTCACGCGCGCACTCGGTGACGTGCCTGGTCGCCCGATGATTTCTAATGAACCGGAGACCTGTCAGGTACCGATCGAATCCAGCGACTATCTGGTCCT
GCTGGCATGTGATGGCATTTCCGATGTGTTTAATGAGCGCGATCTGTATCAACTGGTCGAAGCGTTCGCCAACGATTACCCGGTTGAAGATTATGCCGAACTCTCTCGTTTTATCTGCACGAAAGCCATTGAAG
CGGGCTCTGCCGACAACGTTAGCGTTGTTATTGGTTTTCTCCGCCCACCGCAGGACGTCTGGAAACTGATGAAACATGAATCTGATGATGAAGATAGCGATGTTACGGACGAAGAATGCCAAGTCCCGATTG
AATCTAGCGATTATCTCGTACTCCTCGCCTGCGACGGCATCAGCGACGTATTCAATGAACGTGACCTGTACCAACTCGTGGAAGCATTCGCAAATGACTACCCTGTAGAAGACTACGCTGAACTGAGCCGCTTT
ATTTGTACGAAGGCTATTGAGGCCGGTAGCGCGGATAATGTCTCTGTCGTCATCGGCTTCCTGCGCCCTCCGCAAGATGTATGGAAGCTCATGAAGCATGAGAGCGACGATGAGGACTCCGACGTGACTGAC
GAGGAATGACAGTAAAGGTGGATACGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC
GAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTG
GCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCT
TCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTA
GGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACC
CTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTC
AGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAAC
TGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGA
TTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCA
TTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGC
TGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTC
CCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACA
TCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATAC
CCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTC
ATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAA
AAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAG
CCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGG
ACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGT
GAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCT
GGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACG
GTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGA
ACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTAC
AATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGC
TTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCT
CATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGG
GCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAA
CATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGT
TCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTT
GTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAA
CGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGC
GTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGC
ATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAA
TGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTG
CGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGC
CCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACT
CGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCAC
TCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCG
CGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCC
GGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAG
GCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTG
GAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGC
AGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCT
CTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGG
TTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCT
CATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTA
GAGGATCGAGATCTCGATCCCGCGAAAT
Fig 11. Elution 1 of FtHAP after purification.
Fig 10. Elution 2 of FtHAP after purification
Fig 9. Agarose gel PCR of pmCherry into pGBR22 plasmid
Lane 1 - 1KB ladder, Lane 2 - sample 1, Lane 3 - sample 2, Lane 4 - sample 3, Lane 5 - sample 4, Lane 6 - sample 5, Lane 7 - sample 6, Lane 8 - sample 7, Lane 9 - sample 8
Agarose gel
Fig 8. 1KB DNA ladder used from NEB used in running gels.
Fig 7. Agarose gel of pGBR22 plasmid, primers M13 forward and reverse:
Lane 1 – 1KB latter
Lane 2 – Sample A
Lane 3 – Sample B
Lane 4 – Sample C
Lane 5 – Sample D
Fig 6. Nanodrop measurement 2 of pNIC-Bsa4 plasmid at 230 nm.
Fig 5. Nanodrop measurement 1 of pNIC-Bsa4 plasmid + FABI at 230 nm.
Fig 4.
Transformation of E. coli with 5ng pNIC-BSA4, NEB 5 alpha grown on LB and Kan agar plates.
Fig. 3
Transformation of E. coli with 25ng pNIC-BSA4, NEB 5 alpha grown on LB and Kan agar plates.
Fig. 2
Transformation of E. coli with 25ng pNIC-BSA4, NEB 5 alpha grown on LB and Kan agar plates.
DNA Sequencing of pGBR22 plasmid:
NNNNNNNNNNNNNGGGCGATTGGGCCCGACGTCGCATGCTCCCGGCCGCCATGGCCGCGGGATTTTAGTGATGGTGATGG
TGATGACCGAGCAAAGAGTGGCGTGCAATGGATATTTCACACTGCTCAACAAATGTGTAATCCTTGTTGTGACTGGTTAC
ATCCAGTTTGCGGTCAACATAGTGATACCCTGGCATCCTCACAGGCTTCTTTGCCTTGTAAGTAGATTTGAATTCACACA
AATAGTAACCACCTCCTTCCAACTTCAGAGCCATAAAGTTGTTTCCTATCAGCATTCCATCTCGTGCAAAGAGACGCTCA
GTGTTGGGTTCCCAGCCCTGTGTCTTCTTCTGCATAACAGGTCCATTGGGAGGAAAGTTCACACCAGAGATTTTGACATT
GTAGATGAAACAGTTGCCTTGGATGCTGGAATCATTGCTGACAGTACACACTGCACCATCTTCAAAGTTCATGATCCTCT
CCCATGTATATCCCTCAGGGAATGACTGCTTTACATAATCAGGGATGTCTTCAGGGTACTTGGTGAATGGTATGCTTCCG
TATTGAGACAGTGGTGATAAAATATCCCAAGCAAATGGCAGAGGTCCACCCTTGGTGACAGTGAGCTTTACCGTCTGCTC
CCCCTCGTAAGGCTTTCCTTTTCCATCGCCTTCGACCTCAAAGTAGTGTCCATTGACCGTGCCTGACATATAAACCTTGT
AGGTCATTTGTTTAGCGATCACACTCATGATATTTCTCCTTCAATCAATCAAAATCACTAGTGCGGCCGCCTGCAGGTCG
ACCATATGGGAGAGCTCCCAACGCGTTGGATGCATAGCTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATCA
TGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAA
AGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTNNNAAANCTGTC
NTGNCAGCTGCATTAATGAATCGGCNACNNNCGGGGNANNNGNNGTTTGCGTANTGNNNCTCNTCCGNTNCNCGCTCANT
GANTCNNNNNNCTCNNNCNNNCGGCNNCNGNNNNNNNGNNTCANCNNNNNNNNNNGNNNGNNNNNNNNNTNNNCCNNNNN
NNNGNNNNNACNNNGAANANNNNNNNNNNNNNNCNAANGCNNNNNNNNNAAAGNCNNNNNNNNNNNNNNNNNNTNNNNNN
Nanodrop Spectrophotometer
Fig 1. Nanodrop spectrophotometer measurements 1 and 2 of pGBR22 plasmid blanked using TE buffer.
Kevin - update your page and properly annotate everything. All of your results for the last 2 weeks shoudl be here. Also repeat this PCR. What was the PCR of? - Dr. B
Kevin - good reverse chronological order, but segment your results by each week (We are now on Week 6). Also put dates for each image (roughly) - Dr. B 070913